EndoFree Plasmid Maxi Kit
Xirmadani waxay ku habboon tahay in laga soo saaro 150 - 300 ml oo ah xal bakteeriya ah oo la dhaqay habeenkii, iyadoo la adeegsanayo habka SDS-alkaline lysis ee la hagaajiyay si loo liso bakteeriyada.Soosaarka cayriin ayaa si door ah loogu daraa Scavenger Endotoxin gaar ah waxaana lagu kala saaraa centrifugation si meesha looga saaro endotoxins.Kadibna, xuubabka jel silica ayaa si xushmad leh ugu xidhan DNA-da plasmid ee xalka iyadoo la raacayo xaaladaha milix-sare iyo pH-yar.Tan waxa ku xiga in lagu daro bakhaar lagu dhaqo si looga saaro wasakhda iyo qaybaha kale ee bakteeriyada.Ugu dambeyntii, milix-hooseeya, pH-ga sare-sare ayaa loo isticmaalaa si looga saaro DNA-da plasmid saafiga ah ee xuubka matrixka silikoon.Xuubka jelka silica wuxuu ka shaqeeyaa xuubka xayeysiiska gaarka ah, iyo farqiga qaddarka xayeysiinta ee u dhexeeya tiirka iyo tiirka waa mid aad u yar oo ku celcelintu waa wanaagsan tahay.Phenol, chloroform iyo reageniyeyaasha kale ee sunta ah looma baahna, sidoo kale maaha tillaabooyinka roobabka ethanol.Qalabkan waxa loo isticmaali karaa in si degdeg ah loo soo saaro 0.2 -1.5 mg oo saafi ah koobi sare oo DNA ah, oo leh heerka soo saarista 80% -90%.Xirmadani waxay isticmaashaa qaacido habraac gaar ah oo meesha ka saaraysa endotoxin, waxa ku jira endotoxin aad buu u hooseeyaa saamaynta gudbinta unuguna waa mid aad u fiican.Plasmaid-ka la soo saaray waxa si toos ah loogu isticmaali karaa dheefshiidka ensaymka, PCR, qoraalka in vitro, beddelka, isku xigxiga iyo tijaabooyinka bayooloojiyada kale ee unugyada.
Xaaladaha kaydinta
RNaseA waa in lagu kaydiyaa -30 ~ -15℃ oo lagu raro ≤0℃.
Scavenger Endotoxin waxa lagu kaydin karaa 2 ~ 8℃ hal bil, lagu kaydiyo -30 ~ -15℃ kaydinta mudada dheerwaxaana lagu raray ≤0℃.
Qaybaha kale waa in lagu kaydiyaa heerkulka qolka (15 ~ 25 ℃) oo lagu raro heerkulka qolka.
Qaybaha
| Qaybaha | 10RXNS |
| RNase A | 750 μL |
| Buffer P1 | 75 ml |
| Buffer P2 | 75 ml |
| Buffer P4 | 75 ml |
| Endotoxin Scavenger | 25 ml |
| Buffer PW | 2 × 22 ml |
| Qaaxada TB | 20 ml |
| FastPure DNA Maxi Columns ( Mid kasta oo ku jira 50ml Tube Collection) | 10 |
| Tube Ururinta bilaashka ah ee Endotoxin | 2 × 5 |
RNaseA:10 mg/ml, oo loo isticmaalo in laga saaro RNA.
Buffer P1:Bakteeriyada hakinta joojinta, ku dar RNaseA Buffer P1 kahor isticmaalka koowaad.
Buffer P2:Bakteeriyada lysis kaydinta (ka kooban SDS/NaOH).
Buffer P4:ka takhalusida wax-ka-qabashada.
Qalabaynta Endotoxin:si wax ku ool ah uga saar endotoxin ka soosaaray plasmid cayriin.
Buffer PW:weel dhaqid, ku dar mugga ethanol ee la cayimay ka hor inta aan la isticmaalin.
Qaaxada dhuuban:meel fogaynta.
FastPure DNA Maxi Columns:tiirarka adsorption DNA plasmid.
Tuubooyinka Aruurinta 50 ml:shaandhaynta tuubooyinka ururinta.
Tuubbada Ururinta ee bilaashka ah ee Endotoxin:tuubooyinka aruurinta DNA-da balasmid.
Agabka la diyaariyay
ethanol Absolute, isopropanol, 50 ml tuubooyinka centrifuge wareega-hoose iyo 50 ml endotoxin-freetuubooyinka centrifuge.
Codsiyada
Alaabtani waxay ku habboon tahay soo saarista ballaaran ee plasmids laga bilaabo 150 - 300 ml oo ah xal bakteeriyadahabeen dhaqan.
Habka tijaabada
1. Qaado 150 - 200 ml (aan ka badnayn 300 ml) oo xal bakteeriya ah oo habeen dhaxa lagu beeray ka dibna badhqiyaastii 11,000 rpm (12,000 × g) 1 - 2 daqiiqo.Iska tuur waxyaabaha ka sarreeya oo ururi bakteeriyada.
∆ Marka la ururinayo in ka badan 50 ml oo xal bakteeriya ah, bakteeriyada waxa la soo ururin karaa iyada oo lagu darayo xal bakteeriyada, centrifugation, tuuraya supernatant iyo tallaabooyinka kale ee isla tuubada 50 ml
dhowr jeer.
2. Ku dar 7.5 ml oo Buffer P1 ah (fadlan hubi in RNaseA lagu daray Buffer P1) centrifugetuubo ay ku jirto bakteeriya oo si fiican ugu qas vortex ama pipetting.
∆ Dib-u-soo-kabashada dhammaystiran ee bakteeriyada tallaabadan ayaa muhiim u ah dhalidda, waana in aanay jirin bakteeriyadu kuuskuus ka dib dib-u-joojinta.Haddii ay jiraan bakteeriyayaal bakteeriya ah oo aan si fiican isku dhafanayn, waxay saameyn doontaa lysis-ka, taasoo keenta dhalidda hooseeya iyo nadiifnimada.Haddii OD600 ee xalka bakteeriyada uu yahay 0.65, waxaa lagu talinayaa in 7.5 ml Buffer P1 la isticmaalo marka laga soo saarayo 150 ml oo ah bakteeriyada bakteeriyada;marka OD600 uu yahay 0.75, 8 ml ee Buffer P1 waa in la isticmaalaa oo mugga Buffers P2 iyo P4 waa in si waafaqsan loo beddelaa.Haddii mugga xalka bakteeriyada la kordhiyo ilaa 200 ml, waxaa lagu talinayaa inmugga Buffers P1, P2, iyo P4 ayaa loo kordhiyaa si saami ahaan ah.
3. Ku dar 7.5 ml oo Buffer P2 ah hakinta bakteeriyada ee tallaabada 2 oo si tartiib ah kor iyo hoos ugu qas 6 – 8jeer oo ku balaadhi heerkulka qolka 4 - 5 min.
∆ Si tartiib ah u leexi si aad si fiican isugu walaaqdo.Vortexing waxay sababi doontaa kala qaybsanaanta DNA-da genomic, taasoo keenta jajabyada DNA-da genomic ee plasmid-ka la soo saaray.Waqtigaan, xalku wuxuu noqdaa mid muuqda oo muuqda, oo muujinaya in bakteeriyada si buuxda loo jeexjeexay.Muddada waa in aanay ka badnayn 5 daqiiqo si looga fogaado burburinta plasmids-ka.Haddii xalku aanu caddayn, waxa laga yaabaa inay bakteeriya badan ka dhalatolysis aan dhamaystirnayn, markaa cadadka bakteeriyada waa in si habboon loo dhimaa.
4. Ku dar 7.5 ml oo Buffer P4 ah hakinta bakteeriyada ee tallaabada 3 oo isla markiiba si tartiib ah u leexi 6 - 8 jeer si aad u oggolaato in xalku si buuxda uga takhaluso Buffer P2.Waqtigan xaadirka ah, roobab qulqulaya cadcad waa inay soo baxaan.Centrifuge in ka badan 11,000 rpm (12,000 × g) 10 - 15 daqiiqo, si taxadir leh u tuubiyaha sare ee tuubada 50 ml oo cusubhammiyo roobka cad ee sabaynaya.
∆ Ku dar Buffer P4 oo isla markiiba leexi si aad si fiican isugu walaaqdo.U dhaaf tuubada si ay u istaagto ilaa roobka cad ee si siman loo qaybiyo inta lagu jiro xalka si looga hortago soo saarista roobabka maxaliga ah taas oo saameyn karta dhexdhexaadinta.Haddi aanu jirin labis cad oo soo da'aya ka hor centrifugation iyo supernatant uusan caddayn ka dib centrifugation, tube ayaa noqon kartacentrifuge 5 daqiiqo oo kale.
5. Ku dar 0. 1 jeer mugga (10% mugga sarreeya, qiyaastii 2.2 ml) ee Scavenger Endotoxin ilaa heerka sare ee tallaabada 4 oo u rogo isku qas.Ku rid xalalka qubeyska barafka ama geli baraf la jajabiyey (ama qaboojiyaha qaboojiyaha) 5 daqiiqo ilaa uu xalku ka beddelo turbid oo uu cad yahay (ama weliwax yar turbid), oo marmar isku qas dhowr jeer.
∆ Ka dib marka Scavenger-ka Endotoxin lagu daro dusha sare, ka sarreeya wuxuu noqon doonaa mid qallafsan laakiinKa sarreeya waa in uu noqdaa mid cad (ama wax yar qallafsan) ka dib marka uu qaboojiyo qubeyska barafka.
6. Ka dib markii supernatant la dhigo heerkulka qolka (> 25 ℃) 10 - 15 min, waxay noqon doontaa turbid sidaheerkulkeedu wuxuu kordhiyaa heerkulka qolka.Kadibna mas'uulka sare waa in la rogaa si loo isku daro.
∆ Haddii heerkulka qolku hooseeyo ama aad rabto in aad hoos u dhigto wakhtiga soo saarista, supernatant-ka waxaa lagu duri karaa qubeyska 37 ~ 42 ℃ 5 - 10 daqiiqo talaabada xigtana waxaa la samayn karaa ka dibwaxay noqotaa turbid.
7. Centrifuge supernatant qiyaastii 11,000 rpm (12,000 × g) 10 daqiiqo heerkulka qolka (heerkulka waa in uu ahaadaa>25℃) si loo kala saaro wajiga.Marxaladda biyaha sare waxay ka kooban tahay DNA-da halka lakabka hoose ee saliidda buluuga ah uu ka kooban yahay endotoxin iyo wasakh kale.WareejintaMarxaladda aqueous ee DNA-ka kooban ilaa tuubo cusub iyoiska tuur lakabka saliidda leh.
∆ Heerkulka inta lagu jiro centrifugation waa in uu ka sarreeyaa 25 ℃ maadaama kala qaybsanaanta wejiga waxtarka leh aysan ahayndhaca haddii heerkulku aad u hooseeyo.
∆ Haddii kala-saarku aanu waxtar lahayn, heerkulka centrifugation waxaa lagu hagaajin karaa 30 ℃ iyowaqtiga centrifugation waxaa la kordhin karaa ilaa 15 min.
∆ Ha dhuuqin lakabka saliidda leh ee buluugga ah maxaa yeelay waxaa ku jira endotoxin iyo wasakh kale.
Farsamaynta
Dib u Celinta Dhexdhexaadinta Lysis
◇ Ku dar 7.5 ml Buffer P1
◇ Ku dar 7.5 ml Buffer P2
◇ Ku dar 7.5 ml Buffer P4
Ka saarida Endotoxin
◇Ku dar 0. 1 jeer mugga sare ee Endotoxin Scavenger
Isku-xidhka iyo dhaqidda
◇ Ku dar 0.5 jeer mugga isopropanol
◇ Ku dar 10 ml PW Buffer
◇ Ku dar 10 ml PW Buffer
Ilbaxnimo
◇ Ku dar 1 - 2 ml TB Buffer ama Endotoxin-free ddH2O
